Prokaryotic transposable elements are discrete DNA sequences capable of insertion at single or multiple sites within a prokaryotic genome. Normally, such elements consist of a gene encoding a transposase protein and a transposable cassette comprising a resistance gene flanked by sequences recognized by the transposase protein. Transposition of the transposable cassette into the genome of a host cell (which may, e.g. take place at random or at hot spot sites) occurs via recognition and interaction with the flanking sequences of the transposable cassette by the transposase protein.
Different classes of transposable elements exist. One class comprises i) insertion sequences (IS) which are small (less than 2 kb) DNA fragments encoding transposase proteins or other determinants mediating transposition, and ii) composite transposons, i.e. DNA fragments flanked by two copies of an insertion sequence. The terminal portions of all IS sequences comprises inverted repeat sequences. The transposase protein functions by recognizing these terminal sequences and interacting with the sequences to effect transpositions within the genome.
The second class of transposons is the Tn3 family of tranposons. These transposons encode two products involved in a two-step transposition process: a transposase and a resolvase.
Transposons belonging to this second class have inverted terminal repeats of approximately 35-40 bp.
The third class includes bacteriophage Mu and related phages. Bacteriophage Mu is large relative to other transposons with a genome of 36 kb. Mu encodes two gene products which are involved in the transposition process, a 70 kDa transposase and an accessory protein of approximately 33 kDa. An unusual feature of Mu that distinguishes it from other tranposons is that its ends are not inverted repeat sequences. The Mu transposase has, however, been shown to bind to both ends in an in vitro binding assay.
Transposons have been used extensively for mutagenesis and cloning in gram-positive and gram-negative bacteria: Youngman, P. J., Perkins, J. B., Losick, R. (1983) Genetic transposition and insertional mutagenesis in Bacillus subtilis with Streptococcus faecalis transposon Tn917, Proc. Natl. Acad. Sci. USA, 80, 2305-2309; Youngman, P., Perkins, J. B., Losick, R. (1984), Construction of a cloning site near one end of Tn917 into which foreign DNA may be inserted without affecting transposition in Bacillus subtilis or expression of the transposon-borne erm gene, Plasmid 12, 1-9; Youngman, P. (1985) Plasmid vectors for recovering and expoliting Tn917 transpositions in Bacillus subtilis and other gram positives, p. 79-103 in K. Hardy (ed.), Plasmids: a practical approach, IRL Press, Oxford; Kleckner, N., Roth, J., Botstein, D. (1977) Genetic engineering in vivo using translocatable drug-resistance elements. New methods in bacterial genetics, J. Mol. Biol., 116, 125-159; Wati, M. R., Priest, F. G., Mitchell, W. J. (1990) Mutagenesis using Tn917 in Bacillus licheniformis. FEMS microbiol. Lett., 71, 211-214; Petit, M.-A., Bruand, C., Janniere, L., Ehrlich, S. D. (1990) Tn10-derived transposons active in Bacillus subtilis. J. Bacteriol., 172, 6736-6740.
The latter reference describes pHV1248 and pHV1249, plasmids that are thermosensitive for replication, which carry a transposase gene from Tn10 modified to be expressed in B. subtilis, and sufficient sequences from the IS10 elements of Tn10 flanking a chloramphenicol resistance gene (mini-Tn10) to allow transposition of mini-Tn10 into the B. subtilis chromosome.
Maguin et al. (Maguin, E., Duwat, P., Hege, T., Ehrlich, D, Gruss, A. (1992) New thermosensitive plasmid for gram-positive bacteria, J. Bacteriol. 174, 5633-5638) describe an alternative version of this system.
EP 485 701 discloses the use of transposons for introduction of single copies of a DNA sequence into a prokaryotic cell genome, the transposase protein being encoded in cis.
Slugenova et al., ((1993), Enhanced .alpha.-amylase production by chromosomal integrtation of PTVA1 in industrial strain in B. subtilis, Biotechnology Letters, 15, 483-488) describe the multiple integration of plasmid pTVA1 comprising a modified transposon Tn917 and an .alpha.-amylase gene of interest located outside the transposon. Antibiotic resistance marker genes are present in the resulting strain.
EP 0 332 488 describes a transposition based system for construction of multicopy bacterial strains, i.e. strains comprising multiple copies of a gene of interest, which strains further comprise multiple copies of a selectable marker gene introduced with the gene of interest. The system is exemplified by use of a phage Mu transposon for modification of gramnegative bacteria.
WO 95/01095 describes the use of a minitransposon as a vector for stably tranforming an exogenous gene into a eukaryotic (e.g. animal) chromosome.
Simon, R., Priefer, U., Puhler, A. ((1983), A broad host range mobilization system for in vivo genetic enginering: Transposon mutagenesis in gram-negative bacteria, Bio/Technology, 1, 784-791) describe the use of E. coli specific vectors to transfer transposons into other gram-negative strains by conjugation.
Several of the above described multicopy strains have been produced by integration of a genetic construct comprising the gene of interest and an antibiotic selectable marker and amplifying said construct by culturing the cell in the presence of increasing dosages of antibiotic. Thus, the resulting cell typically comprises a number of antibiotic resistance genes. The presence of such genes are undesirable, in particular from an environmental and a product approval point of view.
Non-antibiotic selection markers have been used for construction of multicopy strains. Herrero, M., de Lorenzo, V., Timmis, K. N. ((1990), Transposon vectors containing non-antibiotic resistance selection markers for cloning and stable chromosomal insertion of foreign genes in gram-negative bacteria, J. Bacteriol., 172, 6557-6567) describe such as system in which herbicide or heavy metal resistances are used as selection markers.
Another alternative to the use of antibiotic resistance markers are described in DE 4 231 764 in which an alternating selection of Thy.sup.- (trimethoprim resistance) and Thy.sup.+ (thy prototrophy) is used for introduction of product genes in Bacillus spp. thereby avoiding the need for selectable markers.
Specific deletion of DNA segments from the chromosomes of bacterial species have traditionally been performed by the methods of homologous recombination (Hamilton, C. H., Aldea, M., Washburn, B. K., Babitzke, P. (1989). New method of generating deletions and gene replacement in Escherichia coli. J. Bacteriol., 171, 4617-4622.; Maguin, E., Duwat, P., Hege, T., Ehrlich, D, Gruss, A. (1992). New thermosensitive plasmid for gram-positive bacteria. J. Bacteriol. 174, 5633-5638.). However, the use of homologous recombination to delete resistance marker genes from strains having multiple, tandem copies of such genes each linked to a copy of the gene of interest is hardly applicable, as homologous recombination would delete also the extra copies of the gene of interest.
The concept of using site specific recombination systems for integration and retrieval of sequences from the bacterial chromosome, using elements from either phage lambda or P1, and some specific methods of achieving this, has been described (Hasan, N., Koob, M., Szybalski, W. (1994), Escherichia coli genome targeting, I. Cre-lox-mediated in vitro generation of ori.sup.- plasmids and their in vivo chromosomal integration and retrieval. Gene, 150, 51-56). The cre-lox system is further described by Abremski, K., Hoess, R., Sternberg, N. (1983), Studies on the properties of P1 site-specific recombination: Evidence for topologically unlinked products following recombination, Cell, 32, 1301-1311. An alternative system is based on the recombination system of the broad-host range plasmid RP4 (Eberl, L., Kristensen, C. S., Givskov, M., Grohmann, E., Gerlitz, M., Schwab, H. (1994), Analysis of the multimer resolution system encoded by the parCBA operon of broad-host-range plasmid RP4, Mol. Microbiol., 12, 131-141)). Stark, W. M., Boocock, M. R., Sherratt, D. J. (1992), Catalysis by site-specific recombinases, Trends in Genetics, 8, 432-439) is a review article on the mechanism of resolvase action. Camilli et al. ((1994), Use of genetic recombination as a reporter of gene expression, Proc. Natl. Acad. Sci. USA, 91, 2634-2638) describe the use of res sites and resolvase from the .gamma..delta. transposon in Vibrio cholera as a permanent, heritable marker of gene expression from a chromosomal gene. The resolution system is not used for excision of marker genes. Chang, L.-K. et al. ((1994, Construction of Tn917as1, a transposon useful for mutagenesis and cloning of Bacillus subtilis genes, Gene, 150, 129-134) describe the plasmids (pE194) containing erm-res-tnpA (transposase)-tnpR (resolvase) samt IR-res-ori colE1-AB.sup.R 1-AB.sup.R 2-IR (pD917; Tn917ac1). The two res sites are there to allow the transposon to function properly, not for excision of intervening DNA.
The broad host range, gram-positive plasmid pAM.beta.1 (Clewell, D. B., Yagi, Y., Dunny, G. M., Schultz, S. K. (1974) Characterization of three plasmid deoxyribonucleic acid molecules in a strain of Streptococcus faecalis: identification of a plasmid determining erythromycin resistance. J. Bacteriol. 117, 283-289) has been described to contain a resolution system, that resolves plasmid multimers into monomers via a site specific recombination event, requiring a specific plasmid encoded enzyme (resolvase) and a site, res, on the plasmid (Swinfield, T.-J., Janniere, L., Ehrlich, S. D., Minton, N. P. (1991). Characterization of a region of the Enterococcus faecalis plasmid pAM.beta.1 which enhances the segregational stability of pAM.beta.1-derived cloning vectors in Bacillus subtilis. Plasmid 26, 209-221; Janniere, L., Gruss, A., Ehrlich, S. D. (1993) Plasmids, pp. 625-644 in Sonenshein, A. L., Hoch, J. A., Losick, R. (eds.) Bacillus subtilis and other gram-positive bacteria: Biochemistry, Physiology and molecular genetics. American society for microbiology, Washington D.C.).
It has been suggested to use a site-specific recombination system to remove a single selectable marker gene from the genome of a bacterial cell. For instance, Dale, et al. ((1991) Gene transfer with subsequent removal of the selection gene from the host genome, Proc. Natl. Acad. Sci. USA, 88, 10558-10562) describe the use of the cre/lox system for removal of markers from transgenic plants and mentions that the use of this system would obviate the need for different selectable markers in subsequent rounds of gene tranfer into the same host. Kristensen, C. S. et al. (1995), J. Bacteriol., 177, 52-58, describe the use of the multimer resolution system of the plasmid RP4 for the precise excision of chromosomal segments (such as marker genes introduced with heterologous DNA) from gram-negative bacteria. It is stated that the system is envisaged to be of interest in the generation of chromosomal insertions of heterologous DNA segments eventually devoid of any selection marker.
WO 95/02058 describes a new transposon (tn5401) from B. thuringiensis containing transposase, resolvase, and res site. The transposon is used in a plasmid which contains B. thuringiensis DNA (e.g. origin and toxin gene) and, flanked by res sites, non-B. thuringiensis DNA (e.g. E. coli origin, selectable marker genes). The plasmid is introduced into B. thuringiensis. Subsequently, a plasmid expressing the resolvase is introduced (e.g. a thermosensitive plasmid containing the entire tranposons--but only used as resolvase donor) whereby the non-B. thuringiensis DNA is excised from the first plasmid.
Conclusions With Respect to the State of the Art
On the basis of the above citations, the following conclusions may be made as to the state of the art:
The insertion of multiple genes of interest by transposition was known, e.g. as described in EP 332 488. However, all strains carrying multiple transposed sequences of interest contain selectable markers.
The removal of markers via site-specific recombination was know from either chromosome or plasmids (cf. Kristensen et al. (1995), Eberl et al. (1994), WO 95/02058). It was known to remove a marker introduced by transposon.
Multicopy strains without presence of heterologous, selectable marker genes were known (DE 4231 764). These strains were constructed by a cumbersome method depending on the use of the Thy marker.
It is an object of the present invention to construct bacterial cells which harbour a stable, fixed and well-defined copy number of one or more genes of interest, without the presence of selectable marker genes in the final strain.